PROJECT SUMMARY More than 98% of the human genome is composed of non-coding sequences. Noncoding genomic elements, such as regulatory elements for gene expression and noncoding RNAs, are increasingly being recognized to play critical roles in cancer biology. However, large-scale unbiased functional characterization of noncoding genomic elements in cancer remains a major challenge. This project proposes the development of a CRISPR-based Version2 Molecular Chipper technology to transform the research of the noncoding genome in cancer. The CRISPR-technology allows the use of programmable small RNAs, or single-guide RNAs (sgRNAs), to modify the genome at specific loci on both alleles. Based on this principle, we recently published the Version1 Molecular Chipper technology that uses standard molecular biology techniques to create dense-tiling sgRNA library for functional mapping of noncoding genomic regions. Compared to the existing method of microarray- based synthesis of sgRNAs, this Version1 technology has proven to be inexpensive, not limited in the total number of sgRNAs, easily adoptable by regular cancer research laboratories, and importantly, powerful for the identification of novel noncoding regulatory elements. However, this Version1 technology has a major weakness in that most of the sgRNAs in the library are not PAM (protospacer adjacent motif)-specific. This weakness renders the library from the Version1 technology being inefficient for functional mapping, and thus prevents this method to be truly transformative. Here we propose a major improvement (Version2) to overcome this weakness, which is expected to increase the efficiency by ~16 fold, to fully unleash the power of the Molecular Chipper technology. We will achieve this through two complementary aims, with the first aim designed to develop the method and the second aim designed to validate the technology through functional mapping of noncoding regulatory elements controlling the expression of a key haploinsufficient tumor suppressor. This proposed Version2 Molecular Chipper technology addresses the existing bottleneck on functional mapping of the noncoding cancer genome. Successful development of this technology will allow its use across wide areas of cancer research, and help the acceleration of research to understand the functions of the noncoding genome in cancer.